The goal is the understanding of the structural features responsible for catalytic specificity and efficiency in yeast triose phosphate isomerase. The method of approach is a combination of site-directed mutagenesis and X-ray structural analysis. In addition, complete kinetic characterization of every mutant will be carried out. The yeast triose phosphate isomerase system is ideal for such purposes, because the reaction is the simplest in all of metabolism, the enzyme is so efficient that it is believed to have reached evolutionary perfection, and the complete free energy profile of the reaction has been determined (Knowles and Albery). Moreover, the enzyme can be crystallized in two distinct forms in the presence and absence of a transition-state analog inhibitor, and both forms have been solved and refined at 1.9 A resolution. The gene coding for the enzyme has been cloned and sequenced, and expressed at high level in E. coli. Several active site mutants have been made and characterized, and the structure of one has already been determined. The characterization, by kinetic and crystallographic means, of those mutants affecting the electrophilic catalysis by the enzyme will be continued. New mutations at other residues will be introduced to test other structural factors that may be important for the catalytic power of the protein. The active site base will be eliminated to provide an inactive anzyme for direct crystallographic study of enzyme-substrate and enzyme-intermediate complexes. Residues in the catalytically-important flexible loop (168-177) will be altered to test theories of loop function and to provide handles for the chemical labelling of the loop with reporter groups. Attempt will be made to alter the orientation and strength of the alpha helix dipoles in the active site, to evaluate their role in binding and catalysis, by introducing helix-disrupting residues at critical positions. Finally, mutations will be made at several other positions where the crystallographic results and sequence conservation imply a possible role in either active site architecture or catalysis: Ser 96, Cys 126, Glu 97, and Asn 10.